time-to-PS Research Articles

Influence of hypertension on cardiac contractile response of human erythrocyte-derived depressing factor in ventricular myocytes.

Erythrocyte-derived depressing factor (EDDF), a novel hypotensive factor purified from human erythrocytes, elicits endothelium-dependent vasorelaxation by reducing intracellular Ca2+ in vascular smooth muscle cells. However, its cardiac response is unknown. This study was designed to examine the cardiac contractile response of EDDF under both normotensive and hypertensive conditions. Ventricular myocytes were isolated from adult male spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) normotensive rats. Mechanical properties were evaluated using an IonOptix MyoCam system and intracellular Ca2+ was measured with fura-2 fluorescence. Myocytes were electrically stimulated to contract at 0.5 Hz. The contractile properties analyzed included peak shortening (PS), time-to-PS (TPS), time-to-90% re-lengthening (TR(90)), maximal velocity of shortening/re-lengthening (+/- dl/dt), fura-fluorescence intensity change (DeltaFFI), and fura-fluorescence decay rate (tau). SHR rats displayed significantly elevated blood pressure. EDDF (10-9-10-4 g/ml) did not affect PS, TPS, TR(90), DeltaFFI and tau but depressed +/- dl/dt at higher doses in WKY myocytes. However, EDDF depressed PS, +/- dl/dt and DeltaFFI, shortened TPS without affecting TR(90) and tau in SHR myocytes. Pretreatment of the myocytes with the nitric oxide synthase inhibitor Nvarpi-nitro-l-arginine methyl ester (l-NAME) did not affect the EDDF-induced inhibition of PS and +/- dl/dt in SHR myocytes but unmasked an EDDF-induced negative response in WKY myocytes. These data indicate that EDDF may participate in the modulation of cardiac contractile function under hypertensive, but not normotensive, conditions. The cardiac depressive effect of EDDF is unlikely due to release of nitric oxide, as suggested in vascular smooth muscles.

Depressed contractile function and adrenergic responsiveness of cardiac myocytes in an experimental model of Parkinson disease, the MPTP-treated mouse

Radiotracer and biochemical studies have shown that patients with Parkinson disease lack functional sympathetic innervation to the heart. The same observation was made in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an experimental model of Parkinson disease. This study examined the mechanical properties, adrenergic receptor level and intracellular Ca 2+ handling in cardiac myocytes isolated from C57/BL6 mice that received either MPTP (30 mg/kg, i.p., twice in 24 h) or vehicle. Mechanical properties were evaluated using an IonOptix MyoCam® system. Myocytes were electrically stimulated at 0.5 Hz. The contractile properties analyzed included peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR 90), and maximal velocities of shortening and relengthening (±d L/d t). Intracellular Ca 2+ handling was evaluated with fura 2. Myocytes from MPTP-treated mice exhibited a depressed PS (85% of normal), normal TPS, prolonged TR 90 (147% of normal), and reduced ±d L/d t (both 79% of normal). These results were correlated with a 67% reduction of β-adrenergic receptor expression in myocardial membranes from MPTP-treated mice when compared to normal. Myocytes from MPTP-treated mice also exhibited a reduced peak of intracellular Ca 2+ sequestration and sarcoplasmic reticulum (SR) Ca 2+ load (55 and 38% of normal, respectively). The resting intracellular Ca 2+ and Ca 2+-transient decay were comparable to the values seen in myocytes from untreated mice. Myocytes from MPTP-treated and untreated mice were equally responsive over a range of stimulation frequencies (0.1, 0.5, 1, 3 and 5 Hz). Response to norepinephrine (1 μM) and isoproterenol (1 μM) was reduced in myocytes from MPTP-treated mice. These results demonstrate substantial cardiac dysfunctions in this model of experimental Parkinson disease, probably due to reduced adrenergic responsiveness and SR Ca 2+ load.

Paradoxical effects of pyruvate on cardiac contractile function under normal and high glucose in ventricular myocytes

Impaired energy fuel supply and metabolism contribute to the cardiac contractile dysfunctions in diabetes. Pyruvate, a metabolic product of glycolysis and an oxidizable fuel in myocardium, has been demonstrated to enhance cardiac contractile function and alleviate hyperglycemic status. The present study was designed to examine the impact of pyruvate supplementation on cardiac mechanical function under normal glucose (NG) and high glucose (HG) conditions. Isolated adult rat ventricular myocytes were maintained in NG (5.5 mM) or HG (25.5 mM) medium for 24 h in the absence or presence of pyruvate (5 mM). Contractile indices were measured with an IonOptix edge-detection system including peak shortening (PS), maximal velocity of shortening/relengthening (±d L/d t), time-to-PS (TPS), time-to-90% relengthening (TR 90), area underneath shortening ( A con) and relengthening ( A relax). Myocytes maintained in HG medium displayed abnormal mechanical function simulating in vivo diabetes. These abnormalities included reduced PS, ±d L/d t, prolonged TPS/TR 90 associated with enhanced A con and A relax compared to NG myocytes. Interestingly, these HG-induced mechanical dysfunctions were completely abolished by co-incubation of pyruvate. However, NG myocytes developed mechanical defects reminiscent of those of HG following co-incubation with pyruvate. These data suggest that pyruvate may paradoxical affect cardiac mechanical function under different glucose settings and therefore warrant caution when applying pyruvate therapeutically.

High glucose induces cardiac insulin-like growth factor I resistance in ventricular myocytes: role of Akt and ERK activation.

Cardiac resistance to IGF-1 occurs in diabetes and is attributed to cardiac dysfunction in diabetes. However, the mechanism of action responsible for cardiac IGF-1 resistance is still unknown. This study was designed to examine the impact of high glucose on IGF-1-induced contractile response and activation of serine-threonine kinase Akt as well as extracellular signal-regulated kinase (ERK1/2) in cardiac myocytes. Isolated adult rat ventricular myocytes were cultured for 12-18 h in a serum-free medium containing either normal (NG, 5.5 mM) or high (HG, 25.5 mM) glucose. Mechanical properties were evaluated using an IonOptix MyoCam system. Myocytes were electrically stimulated at 0.5 Hz and contractile properties analyzed included peak shortening (PS), time-to-PS (TPS) and time-to-90% relengthening (TR(90)). Intracellular Ca(2+)-induced Ca(2+) release was measured as fura-2 fluorescence intensity change (DeltaFFI). Protein levels of total and phosphorylated Akt and ERK1/2, indicators of Akt and ERK1/2 activation, IGF-1 receptors (pro-IGF-1R and IGF-1Ralpha) as well as the glucose transporter GLUT4 were assessed by Western blot. IGF-1 (10(-10)-10(-6) M) elicited a dose-dependent increase in PS and DeltaFFI in myocytes maintained in NG medium. However, IGF-1 induced a negative response on PS and DeltaFFI in HG myocytes. The IGF-1-induced responses in NG or HG myocytes were blunted by the IGF-1 receptor antagonist H-1356. Western blot analysis revealed that IGF-1Ralpha but not pro-IGF-1R was reduced in HG myocytes. While IGF-1 (10(-6) M) upregulated total Akt protein levels in both NG and HG myocytes, it only induced a significant activation of Akt in NG but not HG myocytes. IGF-1 elicited comparable ERK1/2 activation in both NG and HG myocytes. These results suggest that the cardiac IGF-1 resistance in diabetes is likely attributed, at least in part, to reduced IGF-1R and attenuated IGF-1-induced Akt phosphorylation under elevated extracellular glucose.

Impaired cardiac function and IGF-I response in myocytes from calmodulin-diabetic mice: role of Akt and RhoA.

This study characterized the cardiac contractile function and IGF-I response in a transgenic diabetic mouse model. Mechanical properties were evaluated in cardiac myocytes from OVE26 diabetic and FVB wild-type mice, including peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR(90)) and maximal velocity of shortening/relengthening (+/-dL/dt). Intracellular Ca(2+) was evaluated as Ca(2+)-induced Ca(2+) release [difference in fura 2 fluorescent intensity (Delta FFI)] and fluorescence decay rate (tau). Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a, phospholamban (PLB), Na(+)-Ca(2+) exchanger (NCX), GLUT4, and the serine-threonine kinase Akt were assessed by Western blot. RhoA and IGF-I/IGF-I receptor mRNA levels were determined by RT-PCR and Northern blot. OVE26 myocytes displayed decreased PS, +/-dL/dt, and Delta FFI associated with prolonged TPS, TR(90), and tau. SERCA2a, NCX, and Akt activation were reduced, whereas PLB and RhoA were enhanced in OVE26 hearts. GLUT4 was unchanged. IGF-I enhanced PS and Delta FFI in FVB but not OVE26 myocytes. IGF-I mRNA was increased, but IGF-I receptor mRNA was reduced in OVE26 hearts and livers. These results validate diabetic cardiomyopathy in OVE26 mice due to reduced SERCA2, NCX, IGF-I response, and Akt activation associated with enhanced RhoA level, suggesting a therapeutic potential for Akt and RhoA.

Impact of estrogen replacement on ventricular myocyte contractile function and protein kinase B/Akt activation.

Women with functional ovaries have a lower cardiovascular risk than men and postmenopausal women. However, estrogen replacement therapy remains controversial. This study examined the effect of ovarian hormone deficiency and estrogen replacement on ventricular myocyte contractile function and PKB/Akt activation. Nulliparous female rats were subjected to bilateral ovariectomy (Ovx) or sham operation (sham). A subgroup of Ovx rats received estrogen (E(2)) replacement (40 microg. kg(-1). day(-1)) for 8 weeks. Mechanical and intracellular Ca(2+) properties were evaluated including peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR(90)), maximal velocity of shortening/relengthening (+/-dL/dt), fura 2 fluorescence intensity (FFI), and decay rate. Levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), phospholamban (PLB), and Akt were assessed by Western blot. Ovx promoted body weight gain associated with reduced serum E(2) and uterine weight, all of which were abolished by E(2). Ovx depressed PS and +/-dL/dt, prolonged TPS, TR(90), and decay rate, and enhanced resting FFI, all of which, with the exception of TPS, were restored by E(2). Ovx did not alter the levels of SERCA2a, PLB, and total Akt, but significantly reduced Akt activation [phosphorylated Akt (pAkt)], pAkt/Akt, and the SERCA2a-to-PLB ratio. These alterations in protein expression were restored by E(2). E(2) enhanced PS and +dL/dt in vitro, which was abolished by the E(2) receptor antagonist ICI-182780. Ovx reduced myocyte Ca(2+) responsiveness and lessened stimulating frequency-induced decline in PS, both ablated by E(2). These data suggest that mechanical and protein functions of ventricular myocytes are directly regulated by E(2).

Iso-S-petasin, a hypotensive sesquiterpene from Petasites formosanus, depresses cardiac contraction and intracellular Ca2+ transients in adult rat ventricular myocytes.

Petasites formosanus is an indigenous species of the medicinal plant Petasites which has been used to treat hypertension. Both S-petasin and its isoform iso-S-petasin have been shown to be the effective ingredients in P. formosanus. However, their effect on heart function has not been revealed. This study was to examine the effect of iso-S-petasin on cardiac contractile function at the myocyte level. Ventricular myocytes were isolated from adult rat hearts and were stimulated to contract at 0.5 Hz under 1.0 mM extracellular Ca(2+). Contractile properties were evaluated using an IonOptix MyoCam system including peak shortening (PS), time to PS (TPS), time to 90% re-lengthening (TR(90)) and maximal velocity of shortening/re-lengthening (+/-dL/dt). Intracellular Ca(2+) properties were assessed by fura-2 and presented as Ca(2+)-induced Ca(2+) release (CICR) and intracellular Ca(2+) decay. Acute application of iso-S-petasin (10(-7) to 10(-4) M) elicited a concentration-dependent inhibition in PS and CICR, with maximal inhibitions of 51.0% and 31.0%, respectively. Iso-S-petasin also induced a concentration-dependent inhibition of+/-dL/dt without affecting TPS, TR(90), baseline intracellular Ca(2+) level or intracellular Ca(2+) decay. Elevation of extracellular Ca(2+) from 1.0 mM to 2.7 mM significantly antagonized the iso-S-petasin-induced depression in PS and CICR. These results demonstrated a direct depressant action of iso-S-petasin on ventricular contraction, which may work in concert with its antihypertensive action to reduce the cardiac load. The iso-S-petasin-induced decrease in CICR may play a role in its cardiac depressant effect.

Influence of Gender on Intrinsic Contractile Properties of Isolated Ventricular Myocytes from Calmodulin‐Induced Diabetic Transgenic Mice

Diabetes mellitus impairs ventricular function, which itself may be disparately influenced by gender. This study compared the impact of gender on cardiac contractile response in ventricular myocytes from wild‐type FVB and calmodulin‐induced diabetic transgenic (OVE26) mice at young (2 month) and older (11 month) age. Mechanical and intracellular Ca2+ properties of cardiac myocytes were evaluated using an IonOptix MyoCam® system. Diabetic mice of both genders exhibited significantly elevated blood glucose regardless of age. OVE26 myocytes displayed reduced peak shortening (PS) and maximal velocity of shortening/relengthening (±dL/dt), and prolonged time‐to‐PS (TPS) and time‐to‐90% relengthening (TR90), associated with higher resting intracellular Ca2+ levels and attenuated Ca2+‐induced intracellular Ca2+ release compared with the FVB myocytes. Peak shortening and ± dL/dt were smaller in female FVB groups when compared to the age‐matched male counterparts. However, these gender differences were ablated by the diabetic state. No significant gender‐related differences in intracellular Ca2+ handling were noted in either FVB or OVE26 myocytes, with the exception of overt gender differences in OVE26 mice when age was taken into account. Young female OVE26 mice exhibited better‐preserved mechanical function while older female OVE26 mice displayed the worst mechanical function among all four OVE26 groups. In conclusion, our data confirmed impaired cardiac contractile function in diabetes, partially due to altered intracellular Ca2+ handling, in both genders. Mechanical differences existed between genders but were “cancelled off” by diabetic state. Nevertheless, a “female advantage” in ventricular function may still persist in young female diabetic subjects.

IGF-I attenuates diabetes-induced cardiac contractile dysfunction in ventricular myocytes.

Diabetic cardiomyopathy is characterized by impaired ventricular contraction and altered function of insulin-like growth factor I (IGF-I), a key factor for cardiac growth and function. Endogenous IGF-I has been shown to alleviate diabetic cardiomyopathy. This study was designed to evaluate exogenous IGF-I treatment on the development of diabetic cardiomyopathy. Adult rats were divided into four groups: control, control + IGF-I, diabetic, and diabetic + IGF-I. Streptozotocin (STZ; 55 mg/kg) was used to induce experimental diabetes immediately followed by a 7-wk IGF-I (3 mg. kg(-1). day(-1) ip) treatment. Mechanical properties were assessed in ventricular myocytes including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)) and maximal velocities of shortening/relengthening (+/-dL/dt). Intracellular Ca(2+) transients were evaluated as Ca(2+)-induced Ca(2+) release and Ca(2+) clearing constant. Levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), phospholamban (PLB), and glucose transporter (GLUT4) were assessed by Western blot. STZ caused significant weight loss and elevated blood glucose, demonstrating the diabetic status. The diabetic state is associated with reduced serum IGF-I levels, which were restored by IGF-I treatment. Diabetic myocytes showed reduced PS and +/-dL/dt as well as prolonged TPS, TR(90), and intracellular Ca(2+) clearing compared with control. IGF-I treatment prevented the diabetes-induced abnormalities in PS, +/-dL/dt, TR(90), and Ca(2+) clearing but not TPS. The levels of SERCA and GLUT4, but not PLB, were significantly reduced in diabetic hearts compared with controls. IGF-I treatment restored the diabetes-induced decline in SERCA, whereas it had no effect on GLUT4 and PLB levels. These results suggest that exogenous IGF-I treatment may ameliorate contractile disturbances in cardiomyocytes from diabetic animals and could provide therapeutic potential in the treatment of diabetic cardiomyopathy.

Sepsis-induced depressed contractile function of isolated ventricular myocytes is due to altered calcium transient properties.

Chronic peritoneal sepsis in a rodent model produces myocardial dysfunction characterized by decreased rates of ventricular contraction and relaxation in the isolated heart preparation. However, it remains controversial whether the ventricular contractility is altered during sepsis. In the present study, we determined the effect of chronic peritoneal sepsis on the mechanical properties and intracellular Ca2+ handling of cardiac myocytes isolated from septic rats at 24 or 48 h. Mechanical properties were evaluated by use of an IonOptix MyoCam system. Myocytes were electrically stimulated at 0.5 Hz. The contractile properties analyzed included peak shortening (PS), time-to-peak shortening (TPS), time-to-90% relengthening (TR90), and maximal velocities of shortening and relengthening (+/-dL/dt). Intracellular Ca2+ handling was evaluated with fura-2 fluorescent dye. Myocytes obtained from 24-h postseptic animals exhibited a depressed PS (85% of control), normal TPS, prolonged TR90 (147% of control), and reduced +/-dL/dt (both 79% of control). Myocytes from 48-h postseptic animals also exhibited a reduced peak of intracellular Ca2+ sequestration (55% of control), but resting intracellular Ca2+ and Ca2+-transient decay were comparable with the values seen in myocytes from untreated rats. Myocytes from septic and control animals were equally responsive over a range of stimulation frequencies (0.1-5 Hz). Myocytes from septic animals were unresponsive (5% of control) to increase of extracellular Ca2+ (0.5-3 mM). These results demonstrate that sepsis produces substantial deficits in cardiac myocytes function that can be attributed to altered calcium transient properties.

Comparison of cardiac excitation–contraction coupling in isolated ventricular myocytes between rat and mouse

Transgenic animals offer many advantages for physiological study. The mouse is the most extensively utilized mammalian model for gene modification. Isolated ventricular myocytes are pivotal for assessment of cardiac function by allowing direct cellular and environmental manipulation without interference from compensatory mechanisms that may exist in vivo. This study was designed to compare the basic excitation–contraction coupling properties of mouse and rat ventricular myocytes. Cardiac myocytes were isolated from age- and gender-matched mice (FVB and C57BL/6) and rats (Sprague–Dawley (SD) and Wistar). Mechanical and intracellular Ca 2+ properties were measured with an IonOptix SoftEdge system, including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR 90), maximal velocity of shortening and relengthening (±d L/d t), and intracellular Ca 2+ fura-2 fluorescence intensity and decay rate (τ). Resting cell length was variable among the different species or strains. PS from FVB group was significantly higher than the SD group. TPS and TR 90 were significantly shorter in mice. +d L/d t was similar among all groups whereas −d L/d t was significantly faster in the C57BL/6 group compared to the rat groups. Resting intracellular Ca 2+ was lower in mice than in rats, and Ca 2+-induced Ca 2+ release was variable among the four groups. Intracellular Ca 2+ decay was slower in Wistar compared to all other groups. The myocytes from C57BL/6 did not respond to increases in extracellular Ca 2+. Myocytes from the FVB group exhibited a lesser reduction in PS in response to elevated stimulus frequency. These data suggest that inherent differences between strains or species should be taken into consideration when comparing results from these different animal models.

Alpha2-Heremans Schmid glycoprotein, a putative inhibitor of tyrosine kinase, prevents glucose toxicity associated with cardiomyocyte dysfunction.

Diabetes leads to impaired glucose metabolism and insulin signaling in the heart, which may contribute to the development of diabetic cardiomyopathy. Insulin stimulates tyrosine phosphorylation of the insulin receptor and insulin receptor substrates. A two-fold increase in insulin-stimulated tyrosine phosphorylation has been reported in diabetic myocardium. The aim of the present study was to examine the effect of a putative inhibitor of tyrosine kinase phosphorylation, alpha(2)-Heremans Schmid glycoprotein (AHSG), on the mechanical dysfunction under a simulated diabetic environment. Isolated ventricular myocytes from adult rats were maintained for 24 h in either normal glucose (NG, 5.5 mM) or high glucose (HG, 25.5 mM) medium with 10(-7) M insulin, and in the absence or presence of AHSG (50 micro g/ml). Contractile indices analyzed included: peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)) and area underneath shortening and relengthening (Area/PS). Myocytes maintained in HG medium displayed reduced PS and prolonged TPS/TR(90), with enhanced area, compared to the NG myocytes. Interestingly, these HG-induced mechanical dysfunctions were abolished by AHSG. Removal of insulin from the culture medium did not affect the basal myocyte mechanics, but prevented AHSG from completely protecting against the HG-induced mechanical defects (i.e. HG-induced prolongation of TR(90) and area were only partially attenuated by AHSG in the absence of insulin). The present data support the notion of tyrosine phosphorylation in the pathogenesis of diabetic cardiomyopathy, and implicate the therapeutic value of tyrosine kinase phosphorylation inhibitors.

Influence of genetically predisposed diabetes on ethanol-induced depression of cardiac contraction in adult rat ventricular myocytes.

Diabetes mellitus and alcohol (ethanol) intake are two positively correlated major risk factors for cardiovascular abnormalities. However, the interaction of the two on cardiac function is largely unknown. The purpose of the present study was to examine the impact of genetically predisposed diabetes on acute ethanol exposure-induced cardiac contractile depression at the myocyte level. Ventricular myocytes from spontaneously biobreeding diabetes-prone (BBDP) rats and their diabetes-resistant littermates (BBDR) were stimulated to contract at 0.5 Hz. Contractile properties analysed include: peak shortening amplitude (PS), time-to-PS (TPS), time-to-90 % relengthening (TR(90)) and maximal velocities of shortening/relengthening (+/- dL/dt). BBDP rats displayed hyperglycaemia, reduced body weight gain and increased cardiac, hepatic and renal size. Myocytes isolated from BBDP rat hearts exhibited prolonged TPS and TR(90) associated with normal PS and +/- dL/dt, compared with myocytes from the BBDR group. Acute ethanol exposure (80-640 mg dl(-1)) caused a concentration-dependent inhibition of PS in both BBDR and BBDP myocytes. However, the degree of inhibition of PS was significantly reduced in BBDP myocytes compared to that of BBDR myocytes. The maximal inhibition was 52.9 % and 28.4 % in BBDR and BBDP groups, respectively. Ethanol significantly depressed +/- dL/dt in both BBDR and BBDP myocytes. In addition, ethanol did not affect TPS or TR(90) in either the BBDR or BBDP group. Collectively, these results suggest that the ethanol-induced depression in cardiac myocyte contraction may be 'shadowed' by genetically predisposed diabetes.

Cardiac contractile function is enhanced in isolated ventricular myocytes from growth hormone transgenic mice.

Growth hormone (GH) plays a key role in cardiac growth and function. However, excessive levels of GH often result in cardiac dysfunction, which is the major cause of death in acromegalic patients. Transgenic mice with GH over-expression serve as useful models for acromegaly and exhibit impaired cardiac functions using echocardiography, similar to those of human acromegaly. However, the mechanism underscoring the impaired ventricular function has not been well defined. This study was designed to evaluate the cardiac excitation-contraction coupling in GH over-expressing transgenic mice at the single ventricular myocyte level. Myocytes were isolated from GH and age-matched wild-type mouse hearts. Mechanical properties were evaluated using an IonOptix MyoCam system. The contractile properties analyzed included peak shortening (PS), time-to-peak shortening (TPS) and time-to-90% relengthening (TR(90)), and maximal velocities of shortening/relengthening (+/-dL/dt). Intracellular Ca2+ properties were evaluated by fura-2. GH transgenic mice exhibited significantly increased body weights and enlarged heart and myocyte size. Myocytes from GH transgenic mice displayed significantly enhanced PS and+/-dL/dt associated with similar TPS and TR(90) compared with the wild-type littermates. Myocytes from GH transgenic mice displayed a similar resting intracellular Ca2+ level and Ca2+ removal rate but exhibited an elevated peak intracellular Ca2+ level compared with the wild-type group. Myocytes from both groups were equally responsive to increases in extracellular Ca2+ concentration and stimulating frequency. These results suggest that GH over-expression is associated with enhanced contractile function in isolated myocytes and that the impaired cardiac function observed in whole hearts may not be due to defects at the myocyte level.

Impaired cardiac excitation-contraction coupling in ventricular myocytes from Ames dwarf mice with IGF-I deficiency.

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are involved in the regulation of cardiovascular function. GH/IGF-I deficiency is associated with impaired cardiac performance manifested as reduced left ventricular ejection fraction and diastolic filling. This study was to determine the impact of IGF-I deficiency on single cardiac myocyte excitation-contraction (E-C) coupling. Ventricular myocytes were isolated from adult Ames dwarf mice and age-matched wild-type siblings. Dwarf mice are characterized by severe IGF-I deficiency. Mechanical properties were evaluated using a video edge detection system. Myocytes were electrically stimulated at 0.5 Hz. The contractile properties analysed included peak shortening (PS), time to peak shortening (TPS) and time to 90% relengthening (TR(90)), and maximal velocities of shortening/relengthening (+/-d L/d t). Intracellular Ca(2+) transients were evaluated by fura-2 fluorescence microscopy. Dwarf mice exhibited significantly reduced body and heart weights and severely deficient plasma IGF-I. Myocytes from dwarf mice displayed significantly smaller cell lengths (CLs), prolonged TPS/TR(90) and reduced +/-d L/d t compared with the wild-type littermates. The absolute PS was similar although PS/CL was enhanced in the dwarf group. Myocytes from dwarf animals displayed reduced peak intracellular Ca(2+) levels and slowed intracellular Ca(2+) clearing associated with a comparable resting intracellular Ca(2+). Furthermore, myocytes from the dwarf hearts were equally responsive to an elevation in extracellular Ca(2+) and exhibited an augmented stepwise decrease in response to minimal increase in stimulating frequencies compared with those from the wild-type group. These results suggest that deficiency in IGF-I may be directly associated with cardiac E-C coupling dysfunction at the ventricular myocyte level.

Anisodamine inhibits cardiac contraction and intracellular Ca 2+ transients in isolated adult rat ventricular myocytes

Increased cardiac workload often leads to serious complications during cardiac surgery such as pericardiopulmonary bypass. Various agents have been applied to lower peripheral resistance and cardiac workload, one of which, anisodamine, is widely used in Asia. However, the direct action of anisodamine on cardiac contractile property is essentially unknown. This study was designed to examine the influence of anisodamine on ventricular contractile function at the single cardiac myocyte level. Ventricular myocytes from adult rat hearts were stimulated to contract at 0.5 Hz, and mechanical and intracellular Ca 2+ properties were evaluated using an IonOptix Myocam system. Contractile properties analyzed included peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR 90), maximal velocity of shortening/relengthening (±d L/d t), intracellular Ca 2+ fluorescence intensity change (ΔFFI) and decay ( τ). Anisodamine exhibited a concentration-dependent (10 −12–10 −6 M) inhibition in PS and ΔFFI, with maximal inhibitions of 44.7% and 47.2%, respectively. Anisodamine inhibited ±d L/d t, lowered resting FFI but elicited no effect on TPS/TR 90 and τ. Pretreatment with the nitric oxide synthase (NOS) inhibitor N ω-nitro- l-arginine methyl ester ( l-NAME, 100 μM) abolished the inhibitory effect of anisodamine in cell shortening. In addition, anisodamine prevented cholinoceptor agonist carbachol-induced positive cardiac contractile response. This study demonstrated a direct cardiac depressive action of anisodamine at the myocyte level, which may be related to, at least in part, NO production and cholinoceptor antagonism.

Characterization of Contractile Function in Diabetic Hypertensive Cardiomyopathy in Adult Rat Ventricular Myocytes

Diabetes and hypertension both produce myocardial dysfunction that accelerates cardiovascular morbidity and mortality. Coexistence of the two often results in a more severe cardiomyopathy than either process alone. The purpose of this study was to characterize the contractile function of diabetic hypertensive cardiomyopathy at the single myocyte level. Adult spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were made diabetic with a single injection (55 mg/kg) of streptozotocin (STZ). Contractile properties of ventricular myocytes were evaluated, including peak shortening (PS), time-to-peak shortening (TPS), time-to-90% relengthening (TR90) and maximal velocities of shortening/relengthening (±dL/d t). The experimental animals exhibited enlarged heart size, elevated blood glucose and systolic blood pressure. PS was unchanged (SHR), enhanced (WKY-STZ) or depressed (SHR-STZ) compared to control (WKY). Myocytes from all experimental groups displayed prolonged TPS and TR90compared to the WKY group, although only those from the hypertensive groups (SHR, SHR-STZ) were associated with reduced ±dL/d t. Additionally, myocytes from the WKY-STZ but not the SHR or the SHR-STZ groups exhibited impaired responsiveness to increased extracellular Ca2+. Myocytes from the SHR-STZ group displayed a leftward shift of the stimulus frequency-peak shortening response curve compared to the WKY group. These results confirmed observations at the multicellular levels that combination of diabetes and hypertension results in a greater impairment of cardiac contractile function than is seen with either disease alone.

Isolated ventricular myocytes from copper-deficient rat hearts exhibit enhanced contractile function.

Dietary copper deficiency leads to cardiac hypertrophy, cardiac fibrosis, derangement of myofibrils, and impaired cardiac contractile and electrophysiological function. The purpose of this study was to determine whether impaired cardiac function from copper deficiency is due to depressed contractile function at the single myocyte level. Male Sprague-Dawley rats were fed diets that were either copper adequate (5.59-6.05 microg copper/g body wt; n = 11) or copper deficient (0.29-0.34 microg copper/g body wt; n = 11) for 5 wk. Ventricular myocytes were dispersed and mechanical properties were evaluated using the SoftEdge video-based edge-detection system. Intracellular Ca2+ transients were examined using fura 2-acetoxymethyl ester. Myocytes were electrically stimulated to contract at 0.5 Hz. Properties evaluated included peak shortening (PS), time to peak shortening (TPS), time to 90% relengthening (TR90), and maximal velocities of shortening and relengthening (+/-dL/dt). Myocytes from the copper-deficient rat hearts exhibited significantly enhanced PS values associated with shortened TR90 measurements compared with those from copper-adequate rat hearts. The +/-dL/dt values were enhanced and the intracellular Ca2+ transient decay rate was depressed in myocytes from copper-deficient rats. These data indicate that impaired cardiac contractile function that is seen in copper-deficient whole hearts might not be due to depressed cardiac contractile function at the single cell level but rather to other mechanisms such as cardiac fibrosis.

Influence of Ovariectomy on Ventricular Myocyte Contraction in Simulated Diabetes

We studied the effect of ovariectomy (OVX) on cardiac contraction in myocytes maintained under a ‘diabetes-simulated high-glucose’ environment. Female rats were ovariectomized or sham operated (SHAM) and kept for 6 weeks. Isolated myocytes were maintained in a diabetes-simulated high [glucose] medium (HG; 25.5 mM) for 24 h before mechanical properties were measured. Contractile indices analyzed included peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR₉₀), maximal velocity of shortening and relengthening (± dL/dt), intracellular Ca²⁺ fura-2 fluorescence intensity and decay rate (τ). Nitric oxide synthase (NOS) activity was also evaluated. OVX myocytes displayed a longer TR₉₀, slower ± dL/dt, lower fluorescence intensity and higher τ (slower decay rate) when compared to SHAM myocytes. In the SHAM group, HG exerted diabetes-like contractile dysfunctions, including depressed PS, prolonged TR₉₀, reduced fluorescence intensity, higher τ and enhanced NOS activity when compared to myocytes maintained in low [glucose] medium (5.5 mM). Interestingly, the HG- induced mechanical alterations were significantly exaggerated (TPS, TR₉₀ and τ), reversed (PS and NOS) or lost (± dL/dt and fluorescence intensity) in the OVX group. These data suggest that ovarian hormones play a role in the regulation of cardiac contractile function, and may have potentially protective effects against diabetes-associated cardiac dysfunction.

Influence of ovariectomy on ventricular myocyte contraction in simulated diabetes.

We studied the effect of ovariectomy (OVX) on cardiac contraction in myocytes maintained under a 'diabetes-simulated high-glucose' environment. Female rats were ovariectomized or sham operated (SHAM) and kept for 6 weeks. Isolated myocytes were maintained in a diabetes-simulated high [glucose] medium (HG; 25.5 mM) for 24 h before mechanical properties were measured. Contractile indices analyzed included peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening and relengthening (+/- dL/dt), intracellular Ca2+ fura-2 fluorescence intensity and decay rate (tau). Nitric oxide synthase (NOS) activity was also evaluated. OVX myocytes displayed a longer TR(90), slower +/- dL/dt, lower fluorescence intensity and higher tau (slower decay rate) when compared to SHAM myocytes. In the SHAM group, HG exerted diabetes-like contractile dysfunctions, including depressed PS, prolonged TR90, reduced fluorescence intensity, higher tau and enhanced NOS activity when compared to myocytes maintained in low [glucose] medium (5.5 mM). Interestingly, the HG- induced mechanical alterations were significantly exaggerated (TPS, TR90 and tau), reversed (PS and NOS) or lost (+/- dL/dt and fluorescence intensity) in the OVX group. These data suggest that ovarian hormones play a role in the regulation of cardiac contractile function, and may have potentially protective effects against diabetes-associated cardiac dysfunction.